Expression vector for animal cell containing nuclear matrix attachment region of interferon beta

ABSTRACT

The present invention relates to mammalian expression vectors including nuclear matrix attachment region of human interferon β, and more particularly to pPGM-1, pPGM-2 and pPGM-3 including nuclear matrix attachment region of interferon β gene. Those expression vectors confer position independent expression of the introduced foreign gene, thus increasing the frequency of colonies which efficiently express the recombinant protein.

BACKGROUND OF THE INVENTION

[0001] (a) Field of the Invention

[0002] The present invention relates to mammalian expression vectors including a nuclear matrix attachment region of interferon β gene, and more particularly to pPGM-1, pPGM-2, and pPGM-3.

[0003] (b) Description of the Related Art

[0004] Various expression systems including microorganisms, plants, yeasts, insect cells, mammalian cells, etc. have been used to express and obtain target proteins in large quantities to apply to medical and industrial uses. Microorganisms are the easiest systems to use, and microorganism expression systems suitable for various applications have been developed and are commonly used.

[0005] However, microorganism expression systems have some limitations. The most serious limitation is that since protein expression and modification mechanisms (glycosylation, phosphorylation, amidation) of microorganisms differ from those of mammalian cells, even if the same gene is expressed in a microorganism system, the structure or characteristics of expressed proteins is not completely identical to the original protein. Therefore, production of recombinant proteins using microorganism expression systems frequently expresses proteins that are inactivated because modification does not occur after synthesis, or that partially differ in modification or structure even if they are not significantly different in function. In addition, the recombinant protein production process using microorganism expression systems should be accompanied by additional contaminant removal due to contamination of microorganisms, contamination of microorganism endotoxin, etc.

[0006] Meanwhile, mammalian expression systems, although they are the most suitable systems for expressing mammalian proteins, have not been easily industrialized because recombinant protein expression efficiency is low and thus the unit cost of production is high, and the mammalian cell handling process is difficult. Presently used industrial mammalian cell lines include CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney), myeloma, etc., and an expression vector including the gene of interest is transfected into the mammalian cell line to express aimed foreign proteins.

[0007] Mammalian cells maintain various protein modification mechanisms including glycosylation, and protein obtainment and purification processes are easier when proteins are secreted to a culture medium. Most mammalian cells require complex additives such as serum protein, etc. in the culture process, while CHO cells can be cultured in a medium without serum and protein, rendering it the most suitable host for expression of recombinant proteins. In addition, characteristics of CHO cells are well known due to their having been used in many studies, and they have advantages of a high growth rate and that mass suspension culture is possible.

[0008] Generally, in order to express a transgene in a mammalian cell, a vector having a selection marker and a transgene are simultaneously transfected. Transfected cells are cultured and selected in a selection medium. However, expression frequency thereof is very low. One of the reasons is that these transgenes should be integrated in chromosomes of a host cell in mammalian cells contrary to the microorganism system. Additionally, even if stable transfectants are selected, the expression amount is difficult to predict. This is because gene integration positions differ according to cells, and expression aspects differ according to integration positions. Therefore, the copy number of transgenes in mammalian cells and the gene expression amount do not have an explicit correlation therebetween (Grindley et al., 1987, Trends Genet. 3, 16-22; Kucherlapati et al., 1984, Crit. Rev. Biochem. 16, 349-381; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-400). Gene expression in mammalian cells is mostly repressed by a nucleic acid base near the integration position, and thus stably integrated transgenes would often be expressed in a very low level (Eissenberg et al., 1991, Trends Genet. 7., 335-340; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499).

[0009] Usability of nucleic acid factors for protecting transgene expression from position effects has been reported in many systems. As the nucleic acid factors, an insulator factor and a nuclear matrix attachment region (MAR) or scaffold attachment region (SAR), etc. can be used. Although operation mechanisms thereof have not been clarified, when included in transgene constructs, they induce position independent gene expression and the expression amount is determined by the copy number of gene (McKnight, R. A. et al., 1992, Proc. Natl. Acad. US. 89, 6943-6947).

[0010] Kalos et al. have combined the MAR factor of the human apolipoprotein B gene with a minimal promoter transgene construct and induced gene expression in mammalian cells to increase expression of the transcript by about 200 times (Kalos et al., 1995, Mol. Cell. Biol. 15, 198-207). Similarly, it has been reported that the MAR factor of the chicken lysozyme A gene and the SAR factor of human interferon β, etc. confer position-independent transgene expression in vertebrates (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Klehr et al., 1991, Biochemistry 30, 1264-1270). However, no attempt to apply the MAR/SAR factor to substantially increase protein production in CHO cell lines has been reported, nor has industrial profit been identified.

SUMMARY OF THE INVENTION

[0011] Accordingly, it is an object of the present invention to provide a vector which confers increased foreign protein expression efficiency in mammalian cells.

[0012] It is another object of the present invention to provide a vector for position independent expression of foreign proteins.

[0013] It is still another object of the present invention to provide a vector comprising a nuclear matrix attachment region of interferon β gene for increasing the frequency of foreign protein expression and the amount of foreign protein expressed in mammalian cells.

[0014] It is further object of the present invention to provide a vector comprising multiple cloning sites to facilitate the cloning of transgenes.

[0015] It is still further object of the present invention to provide a vector capable of expressing foreign proteins in mammalian cells.

[0016] In order to achieve these objects, the present invention provides mammalian expression vectors comprising a nuclear matrix attachment region of interferon β gene, a promoter, and a transcription terminator.

[0017] The present invention also provides mammalian cells transfected with the expression vectors in which target genes are introduced.

[0018] The present invention also provides a method for production of proteins comprising the steps of introducing transgenes into mammalian expression vectors and introducing the vector into mammalian cells to express proteins from the transgenes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows a structure of a vector constructed by modifying a pSV-β-gal vector in order to construct the expression vector of the present invention.

[0020]FIG. 2 shows the β-gal staining method which demonstrates the increase in the frequency of positive cells by nuclear matrix attachment region of interferon β gene.

[0021]FIG. 3 shows frequency of positive cell lines that express β-gal when amplifying introduced genes by adding MTX

[0022]FIG. 4 shows β-gal expression frequency and expression amount in a G418 selection medium.

[0023]FIG. 5 shows β-gal expression frequency and expression amount in a DHFR selection medium.

[0024]FIG. 6 shows β-gal expression frequency and expression amount in the presence of a CMV promoter.

[0025]FIG. 7 shows the structure of the pPGM-1 expression vector of the present invention.

[0026]FIG. 8 shows the map of the pPGM-2 expression vector of the present invention and a base sequence of multiple cloning sites.

[0027]FIG. 9 shows the map of the pPGM-3 expression vector of the present invention.

[0028]FIG. 10 shows results of analyzing expression titer of a human growth hormone expression cell line prepared using pPGM-1, by Western Blot.

[0029]FIG. 11 shows results of analyzing expression titer of an interferon β expression cell line prepared using pPGM-1, by Western Blot.

DETAILED DESCRIPTION AND THE PREFERRED EMBODIMENTS

[0030] The present inventors invented optimum expression vectors for overcoming position-specific expression inhibition effect (hereinafter referred to as “position effect”) and improving gene expression amounts in transgene expression in mammalian expression systems.

[0031] The mammalian expression vector of the present invention further added a useful base sequence to the existing expression vectors. The useful base sequence protects transgene expression from position effects of the host cell chromosome, and increases transgene expression in CHO (Chinese hamster ovary) or BHK (Baby hamster kidney) cells, etc., commonly used as commercial mammalian cells. The useful base sequence is preferably the nuclear matrix attachment region (MAR) or saffold attachment region (SAR).

[0032] The MAR/SAR factor is preferably selected from a group consisting of chicken lysozyme 5′ MAR (Phi-Van, L. and Stratling, W. H., 1996, Biochemistry 35, 10735-10742, Gene bank #: X98408), chicken phi α globin 5′MAR (Kraevskii, V. A. et al., 1992, Mol. Biol. 26, 672-678, Gene bank #: X64113), CHO DHFR intron MAR (Kas, E. and Chasin, L. A., 1987, J. Mol. Biol. 198, 677-692, Gene bank #: X06654), human HRRT intron MAR (Sykes, R. C. et al., 1988, Mol. Gen. Genet. 212, 301-309, Gene bank #: X07690), human CSP-B gene flanking SAR (Hanson, R. D. and Ley, T. J., Gene bank #: M62716), and human interferon β gene flanking SAR (Mielke, C. et al., 1990, Biochemistry 29, 7475-7485, Gene bank #: M83137).

[0033] Influences of the MAR/SAR factor on transgene expression were analyzed by integrating 6 kinds of MAR/SAR factors into pSV-β-gal/ver I or pSV-β-gal/ver II and examining expression aspects of β-gal genes.

[0034] Six kinds of MAR/SAR factors were respectively integrated into upstream of a promoter of pSV-β-gal/ver I or pSV-β-gal/ver II to complete a test vector. The test vector was introduced to CHO DG44 cells to prepare a transfected cell line, and β-gal expression frequency and expression amount were measured.

[0035] The recombinant vector pSBβ/I including interferon β MAR factor was compared with pSV-β-gal as a control. The number of colonies increased by about 3 times in a G418 medium, and frequency of positive cell lines expressing β-gal increased to 71% compared to 35% of the control, thus the number of positive cell lines increased by 6 times.

[0036] In addition, when pSVβ/I transfected cell lines were selected in a DHFR selection medium, the number of positive colonies that were produced in initial selection medium not including nucleoside increased by 10 times. When MTX was added to a culture medium to induce transgene amplification, the β-gal positive colony rate of the pSVβ/I transfected cell line was 90% or more of total colonies, and the number of positive cells increased to about 7 times as much as the control PSV-β-gal (FIG. 3).

[0037] Accordingly, the interferon β MAR factor of the present invention increases expression efficiency of transgenes and selection marker genes to largely enhance growth of transfected cell lines in selection medium, and shortens periods required for selecting transfected cell lines in selection mediums.

[0038] The present invention also provides mammalian expression vectors including a nuclear matrix attachment region of interferon β at a upstream of promoter 5′. The mammalian expression vectors are preferably pPGM-1, pPGM-2, and pPGM-3. The promoter is preferably selected from a group consisting of SV40 promoter, CMV (cytomegalovirus) promoter, and MMTV (Mouse Mammary Tumor Virus) promoter.

[0039] The pPGM-1 vector contains a 5409 bp including a nuclear matrix attachment region of the human interferon β gene, a SV40 virus promoter, and multiple cloning sites (MCS), and it can express various genes by integrating transgenes in multiple cloning sites. Multiple cloning sites include HindIII, NheI, NotI, and XhoI restriction sites. The base sequence of the pPGM-1 vector is listed as SEQ ID NO: 1 in the sequence listing program, and major factors included in the vector and their positions are shown in Table 1 and FIG. 7. The pPGM-1 vector was deposited with the Korean Culture Collection of Microorganisms under deposition no. KCCM 10232. TABLE 1 Sequence No. Functions   1-413 Initial promoter and enhancer of SV40 virus  414-448 Multiple cloning sites (MCS)  449-584 Small T antigen of SV40 virus 1194-2054 β-lactamase: Amp^(R) gene 3225-5397 Nuclear matrix attachment region of interferon β gene

[0040] The pPGM-2 vector, which modifies multiple cloning sites of the pPGM-1 vector, includes a nuclear matrix attachment region of the interferon β gene, a SV40 promoter, and multiple cloning sites. The base sequence of the pPGM-2 is listed as SEQ ID NO: 2, and the structure of the vector is shown in FIG. 8 and Table 2. The multiple cloning sites of the pPGM-2 vector include NheI, PmeI, AfIII, BamHI, BstXI, EcoRV, NotI, XhoI, XbaI, ApaI, PmeI, and MluI restriction sites. The pPGM-2 vector was deposited with the Korean Culture Collection of Microorganisms under deposition no. KCCM 10338. TABLE 2 Sequence No. Functions   1-413 SV40 promoter  426-445 T7 promoter  446-579 Multiple cloning sites (MCS) 1331-2191 β-LACTAMASE: Amp^(R) gene 3361-5534 Nuclear Matrix Attachment Region of interferon β gene

[0041] The pPGM-3 vector which substitutes the SV40 promoter of the pPGM-1 vector with the CMV (Cytomegalovirus) derived promoter consists of 5601 bp. The base sequence thereof was shown as SEQ ID NO: 3, and the structure is shown in FIG. 9 and Table 3. The pPGM-3 vector was deposited with the Korean Culture Collection of Microorganisms under KCCM 10339. TABLE 3 Sequence No. Functions   1-611 SV40 promoter  612-640 Multiple cloning sites (MCS) 1396-2246 β-lactamase: Amp^(R) gene 3417-5589 Nuclear Matrix Attachment Region of interferon β gene

[0042] The pPGM-1 vector, pPGM-2 vector, and pPGM-3 vector of the present invention are superior to common vectors in number of positive cells expressing transgenes, frequency, and expression amount. Therefore, the pPGM-1 vector, pPGM-2 vector, and pPGM-3 vector of the present invention can be used for expressing and producing transgenes in mammalian cells, and they can produce recombinant proteins such as enzyme and cytokine.

[0043] In addition, the present invention introduces a transgene into a vector including a nuclear matrix region of the interferon β gene, and introduces it into a mammalian cell to provide a transfected cell line. The transfected cell line is selected by culturing in a medium to which a selection marker or MTX is added. A transgene is a gene encoding all kinds of proteins capable of being expressed as recombinant proteins. Representative examples include insulin, cytokine (interleukin, tumor necrosis factor, interferon, colony stimulation factor, chemokine, etc.), erythropoietin, etc. The transfected cell line can be produced by a common method.

[0044] In addition, the present invention provides a method of producing proteins comprising: introducing a transgene into a vector including a nuclear matrix attachment region of the interferon β gene, and introducing the vector into a mammalian cell to express proteins from a transgene. The vector is preferably selected from a group consisting of pPGM-1 (KCCM 10232), pPGM-2 (KCCM 10338), and pPGM-3 (KCCM 10339). The mammalian cell can be any mammalian-derived cell, and it is preferably CHO (Chinese hamster ovary).

[0045] As described above, the pPGM-1 vector, pPGM-2 vector, and pPGM-3 vector of the present invention improve repression of transgene expression caused by surrounding bases and increase expression. In addition, the expression vector of the present invention can be effectively applied to mass production of industrially useful proteins, and it can produce recombinant proteins having the same structure and functions as original proteins of higher animals.

[0046] The present invention will be explained in further detail with reference to the following Examples and Comparative Examples. However, these are to illustrate the present invention and the present invention is not limited to them.

EXAMPLE 1 Cloning of MAR or SAR Factor

[0047] Genomic DNA was isolated from human G-2 cells using a Wizard Genomic DNA purification kit (Promega, U.S.A.). 10 μg of DNA were cut with ClaI, SmaI, XbaI, and XhoI restriction enzymes to use in the PCR process. DNA to be used in PCR was separated and purified from a CHO chicken cell line and a chicken embryo by a similar method.

[0048] 200 ng of the isolated genomic nucleic acid was used as a template, and 25 pmole of each primer, 0.5 mM of dNTP, and ExTaq polymerase (Takara Shuzo Co., Japan) were added to carry out PCR. The base sequence of primer used for each MAR/SAR factor and size of DNA fragment obtained by PCR are as shown in Table 4. PCR was carried out using a GeneAmp PCR system 9600 (Perkin-Elmer Corp. U.S.A.), and PCR conditions are as shown in Table 5. TABLE 4 SEQ ID MAR/SAR factor Primer NO: Size Chicken lysozyme 5′-GGA TCC ATA ATA TAA CTG TA-3′ 4 1668 bp 5′MAR 5′-AAG CTT AAA AGA TTG AAG CA-3′ 5 Chicken phi α 5′-AAG CTT TTA ACC AAC AAA AA-3′ 6  619 bp globin 5′MAR 5′-CTG CAG ACC TAA CCT GTC AC-3′ 7 CHO DHFR intron 5′-TAT ACG TGA ATA GTT TTT CT-3′ 8  549 bp MAR 5′-GAG TTG GAA CTG AGA AGT TC-3′ 9 Human HPRT 5′-AAG CTT GGT CAA GA TGG TG-3′ 10  580 bp intron MAR 5′-GCT GGG CGT GGT GGT GCC TG-3′ 11 Human CSP-8 5′-GGA TCC GAT TCT CCT TGA TG-3′ 12 1233 bp gene SAR 5′-GAA TTC AAA CAA CTC AAT AG-3′ 13 Human interferon 5′-GAA TTC AGC AAG GTC GCC AC-3′ 14 2174 bp β SAR 5′-TTG TAT CAA CTT TCT ACA AT-3′ 15

[0049] TABLE 5 Step Conditions Cycle 1 94° C., 2 min 1 2 94° C., 40 sec; 65° C., 40 sec; 72° C., 40 sec 2-31 3 72° C., 10 min 32

[0050] Fragments of chicken lysozyme 5′ MAR (Gene bank #: X98408, hereinafter referred to as “lyso MAR”), chicken phi α globin 5′ MAR (Gene bank #: X64113, hereinafter referred to as “phi-a MAR”), CHO DHFR intron MAR (Gene bank #: X06654, hereinafter referred to as “DHFR MAR”), human HPRT intron MAR (Gene bank #: X07690, hereinafter referred to as “HPRT MAR”), human CSP-B gene flaking SAR (Gene bank #: M62716, hereinafter referred to as “CSP-B MAR”), and human interferon β gene flanking SAR (Gene bank #: M83137, hereinafter referred to as “interferon β MAR”) were separated by the above-explained method.

[0051] The PCR product was subcloned in pT7blue (R) (Novagene, U.S.A.) or a pCR 2.1 (Invitrogen, U.S.A.) vector. HPRT MAR, DHFR MAR, and lyso MAR were subcloned in a pT7blue (R) vector, and phi-a MAR, CSP-B MAR, and interferon β MAR were subcloned in PCR 2.1.

EXAMPLE 2 Construction of pSV-β-gal/ver I and pSV-β-gal/ver II Vectors

[0052] In order to effectively clone multiple MAR/SAR factors subcloned in the pT7blue (R) or pCR 2.1 vector at upstream of a SV40 promoter of a pSV-β-gal (hereinafter referred to as ‘pSVβ’) vector, modified pSV-β-gal/ver I and PSV-β-gal/ver II vectors were constructed (FIG. 1).

[0053] (1) Construction of pSV-β-gal/ver I

[0054] PCR was carried out with pSVβ as a template with primers of SEQ ID NO: 16 and SEQ ID NO: 17 to obtain a fragment including the SV40 promoter. The fragment was cut with SpeI and HindIII enzymes, and a 443 bp of DNA fragment including SV40 was purified with a Geneclean III kit (Bio 101, U.S.A). It was subcloned in a opened pBluescript SK (+) vector (Stratagene, U.S.A.) with SpeI and HindIII enzymes to construct pBS/SV40 I.

[0055] The pBS/SV40 I was digested with ScaI and HindIII enzymes to separate a 1240 bp DNA fragment, and it was subcloned in pSVβ treated with the same enzymes to complete a pSV-β-gal/ver vector.

[0056] (2) Construction of pSB-β-gal/ver II Vector

[0057] A pSVβ was cut with EcoRI and HindIII to obtain a 420 bp fragment by the above-explained method, and the fragment was inserted into a pBluescript SK (+) vector opened with the same enzymes to construct a pSV-β-gal/ver II vector.

EXAMPLE 3 Construction of a Vector Including MAR Factor and β-gal Gene

[0058] MAR factors were separated from pT7blue(R)/HPRT MAR, bT7blue(R)/DHFR MAR, pT7blue(R)/lyso MAR, pCR 2.1/phi-a MAR, pCR 2.1/CSP-B MAR, and pCR 2.1/interferon β MAR, and cloned in pSVβ/I or pSVβ/II.

[0059] In a pSVβ/I, phi-a MAR and HPRT MAR (human HPRT intron MAR) were subcloned using SpeI/SmaI, and interferon β MAR and lyso MAR were subcloned using ApaI/SpeI. Constructed vectors were pSV-β-gal/phi-a MAR, pSV-β-gal/HPRT MAR, pSV-β-gal/interferon β MAR (hereinafter referred to as ‘pSVβ/I’), and pSV-β-gal/lyso MAR.

[0060] Additionally, DHFR MAR and CSP-B MAR were subcloned in pSV-β-gal/ver II at BamHI/Xba I restriction site. Constructed vectors were pSV-β-gal/DHFR MAR and pSV-β-gal/CSP-B MAR respectively.

EXAMPLE 4 Construction of pCMVβ and pCMVβ/I

[0061] A SpeI-XhoI fragment including a MAR factor was deleted from a pSVβ/I vector and the vector was self-ligated. After cleavage by ApaI and HindIII, a CMV promoter fragment was inserted to construct a pCMVβ vector. The CMV promoter was amplified by PCR with primers of SEQ ID NO: 18 and SEQ ID NO:19 in a pcDNA3.1 vector. The sequences of SEQ ID NO: 18 and SEQ ID NO:19 include a ApaI or HindIII restriction site.

[0062] Also, a pSVβ I vector was digested with ApaI and HindIII restriction enzymes and a CMV promoter fragment was integrated therein to construct a pCMVβ/I vector.

[0063] Namely, pCMVβ is a vector without the MAR factor, and pCMVβ/I is a vector having the MAR factor.

EXAMPLE 5 Measurement of Gene Expression of pSVβ and pSVβ/I

[0064] (1) Transfection

[0065] A CHO cell line DG44 deficient of the DHFR gene was cultured in a MEM-α medium (α-Minimum Essential Medium, Gibco BRL) to which 10% serum (fetal bovine serum) was added. 2×10⁵ cells were inoculated on a 60 mm plate together with 3 ml of medium and cultured in 5% CO₂ incubator at 37° C. overnight, until transfection occurred.

[0066] Transfection was carried out by a liposome method. A pSV2Neo or pDCH1P vector having selection maker gene was co-transfected with each test vector in a mole ratio of 100:1, and a pSVβ vector was used as a control to perform the same experiment. 2 μg of each vector were mixed with lipid surfactant, DOSPER (BOEHRINGER Mannheim, German) and serum-free MEM-α medium and reacted at room temperature for 45 minutes, it was added to a rinsed cell together with a medium to culture for 5 hours, and then the reaction liquid was removed. Culture was carried out in MEM-α medium including 10% FBS and G418 (500 μg/ml) for 2 weeks in order to select the transfected cell line using a Neo gene, and culture was carried out in MEM-α medium not including a nucleoside for 2 weeks in order to select a DHFR gene, and β-gal expression was analyzed.

[0067] (2) Measurement of Frequency of β-gal Expression Positive Cell Line

[0068] Positive cells expressing β-gal were selected.

[0069] Cells cultured on a 100 mm plate were treated with a fixing agent (2% formaldehyde and 0.2% glutaraldehyde) at 4° C. for 10 minutes. They were washed with 1×PBS twice, and X-gal (1 mg/ml) was added to react them at 37° C.

[0070] A β-gal expression cell can be easily identified because it decomposes X-gal and shows a blue color. Positive cells were separated with a trypsin-EDTA solution, and the number of cells was measured with a hematocytometer.

[0071]FIG. 2 is a photo of cells treated with X-gal after being transfected with pSVβ or pSVβ/I in order to identify β-gal expression cells. (a) shows cells transfected with pSVβ, and (b) shows cells transfected with pSVβ/I. A positive cell line showing a blue color of PSVβ/I transfected cell line is remarkable in the upper plate photo.

[0072] After transfection, the transfected cell line was selected using a Neo gene or a DHFR gene, and the number of positive cells expressing β-gal were measured.

[0073] Table 6 shows the number of β-gal positive cells of DG44/PSVβ or DG44/PSVβ/I selected using the Neo selection factor. Table 7 shows the number of β-gal positive cells of DG44/pSVβ or DG44/pSVβ/I selected using the DHFR selection factor. TABLE 6 Total cell Positive Cell Negative Cell Positive Cell number number number Frequency (%) DG44/pSVβ 222 145 77 34.68 DG44/pSVβ/I 659 191 468 71.02

[0074] TABLE 7 Total Cell Positive Cell Negative Cell Positive Cell number number number Frequency (%) DG44/pSVβ 120 105 15 12.50 DG44/pSVβ/I 350 200 150 42.86

[0075] The total number of colonies and frequency of positive colonies increased, and the number of positive colonies remarkably increased, when the transfected cell lines were selected by culturing in selection maker existing media.

[0076] In addition, the transfected cell line (DG44/pSVβ, DG44/pSBβ/I) was adapted to a MTX-added medium to induce gene amplification. The β-gal expression frequency of the transfected cell line adapted to MTX 10 nM and 50 nM was measured. FIG. 3 is a graph measuring activity of the β-gal of MTX-adapted transfected cell line. 90% or more of the DG44/PSVβ/I transfected cell line selected in MTX expressed β-gal, and produced 7 times as many positive colonies as the control (pSVβ).

[0077] Accordingly, the interferon β MAR factor further enhanced expression activity of the SV40 promoter to increase frequency of positive cell lines by 6-10 times. A common transfected cell line preparation process requires that transfected cell lines should be selected from a selection medium for a very long period. However, it is expected that the MAR factor will sharply shorten the period of time required for development of transfected cell lines. In addition, the MAR factor affects expression of selected genes as well as target genes to largely enhance growth of recombinant cell lines in the selection medium.

[0078] (3) β-gal Activities of DG44/pSVβ and DG44/pSVβ/I

[0079] β-gal activity of the same cell as used to measure frequency of the β-gal expression positive cell line was measured.

[0080] A transfected cell line was cultured for 48 hours and washed with 1×PBS twice. 1 ml of STE (0.1M NaCl, 10 mM Tris-Cl (pH 8.0) and 1 mM EDTA (pH 8.0) solution were added thereto and it was put on ice. Cells were scraped with a scraper, moved to an effendorf tube, centrifuged at 4° C. at 14000 rpm for 40 seconds to remove supernatant, and 100 μl of 0.25 M Tris-Cl (pH 7.5) was added to mix with the cells. A process for freezing under liquid nitrogen and immediately melting at 37° C. was repeated 5 times. The pulverized cells were centrifuged at 4° C. at 12000 rpm for 10 minutes and supernatant was moved to a new tube to analyze it.

[0081] 30 μl of cell extracts, 3 μl of 100× magnesium solution (0.1 M MgCl₂, 4.5 M β-mercaptoethanol), 66 μl of 1×ONPG (4 ml/ml in 0.1 M sodium phosphate (pH 7.5)), and 201 μl of 0.1 M sodium phosphate (pH 7.5) were mixed well, and reacted at 37° C. until the color changed to yellow. After adding 500 μl of 1 M Na₂CO₃, absorbance was measured at 420 nm.

[0082] The protein amount in solution was measured by the bicinchoninic acid (BCA) method (Smith et al., 1985, Anal. Biochem. 150, 76-85).

[0083]FIG. 4 is a graph measuring β-gal activity of DG44/pSVβ or DG44/pSVβ/I selected with the Neo selection maker. FIG. 5 is a graph measuring β-gal activity of DG44/pSVβ or DG44/pSVβ/I selected with the DHFR selection maker.

[0084] When the MAR factor existed in the expression vector, β-gal activities increased 7.5 times (FIG. 4) and 32 times (FIG. 5), respectively. This indicates that, considering the frequency of positive cells, expressed β-gal protein activity per positive cell substantially increased.

EXAMPLE 6 Measurement of β-gal Expression of pCMVβ and pCMVβ/I

[0085] Each of pCMBβ and pCMVβ/I was transfected into DG44 by the same method as in Example 5, and number of β-gal positive cell lines and activities were measured.

[0086]FIG. 6 is a graph showing β-gal activities of DG44/pCMVβ and DG44/pCMVβ/I, and Table 8 shows the number of β-gal positive cells of DG44/pCMVβ and DG44/pCMVβ/I. TABLE 8 Total Cell Positive Cell Negative Cell Positive Cell number number number Frequency (%) DG44/pCMVβ 180 147 33 18.33 DG44/pCMVβ/ 326 79 247 75.77 I

[0087] The MAR factor increased frequency of positive cell lines from 18.33% to 75.77% even under a CMV promoter, and it increased the total number of colonies by about 1.8 times and the number of positive colonies by about 7.5 times. Therefore, it is suggested that the effects of interferon β MAR factor are not limited to the SV40 promoter, and that the MAR factor of the present invention can be used in various promoter including SV40, CMV promoter, and other promoters.

EXAMPLE 7 Construction pPGM-1 Vector

[0088] A β-gal site was deleted from a pSVβ/I vector and multiple cloning sites (MCS) were insertedtroduced to construct a pPGM-1 vector with the genetic map of FIG. 7.

[0089] A HindIII-BamHI fragment including a β-gal gene was removed from a pSVβ/I vector, and 160 bp of a HindIII/BamHI fragment of pMSG (KCCM 10202) was inserted. The pPGM-1 vector was deposited with the Korean Culture Collection of Microorganisms and assigned deposit No. KCCM 10232.

EXAMPLE 8 Construction of pPGM-2 Vector

[0090] A pPGM-1 vector was inserted with multiple cloning sites to construct a pPGM-2 vector.

[0091] A HindIII-BamHI fragment of a pSVβ vector was substituted with a pMSG (KCCM 10202) derived corresponding fragment, and the vector was opened with an EcoRI enzyme to integrate an interferon MAR factor. It was treated with NheI and XhoI enzymes and a fragment including multiple cloning sites and a T7 promoter was ligated. The fragment was prepared using a PCR primer (Sequence No. 20 and Sequence No. 21) comprising AvrII and SalI restriction site.

[0092]FIG. 8 shows the map of the pPGM-2 vector and base sequence of multiple cloning sites, and the vector was deposited with the Korean Culture Collection of Microorganisms and assigned deposit No. KCCM 10338.

EXAMPLE 9 Construction of pPGM-3 Vector

[0093] A HindIII-BamHI fragment of pSVβ-1 vector was substituted with a pMSG (KCCM 10202) derived corresponding fragment. The vector was digested with EcoRI and NheI to delete a promoter and substitute it with a CMV promoter, and it was opened with an EcoRI enzyme to ligate an interferon MAR factor. The structure of the constructed pPGM-3 vector is shown in FIG. 9, and its deposit No. is KCCM 10339.

EXAMPLE 10 Production of Recombinant Protein Using pPGM-1 Vector

[0094] The pPGM-1 vector was digested with NheI and XhoI enzymes and integrated genes were ligated. The genes were cDNA encoding human growth hormone (Gene Bank #: E01424) or human interferon β (Gene Bank #: V00534).

[0095] The constructed expression vector was transfected into CHO DG44 cells and a high expression transfected cell line was selected while increasing the concentration of MTX added to a medium. Expression titer was measured by Western Blot, as shown in FIGS. 10 and 11.

[0096] 1.08×10⁵ cells were put in a 12-well plate and cultured for 48 hours, and the medium was pooled to carry out Western Blot. Lane 1 is 25 ng of positive control protein, and lanes 2-5 are culture solutions derived from different colonies selected. The expression rate for human growth hormones was measured at about 20 μg/ml/day, and the expression rate for interferon β was approximately 15 μg/ml/day.

1 21 1 5409 DNA Artificial Sequence pPGM-1 vector 1 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 60 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 120 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 180 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 240 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 300 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 360 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg 420 ctagcggccg cagatctgtt aactcgagaa cttgtttatt gcagcttata atggttacaa 480 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 540 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcggcatgc aagctggcac 600 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 660 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 720 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta 780 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 840 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 900 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 960 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 1020 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 1080 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1140 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 1200 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 1260 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1320 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1380 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 1440 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1500 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1560 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1620 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1680 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 1740 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1800 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1860 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1920 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1980 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 2040 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 2100 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 2160 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2220 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2280 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2340 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2400 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2460 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2520 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2580 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2640 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2700 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2760 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2820 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2880 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2940 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 3000 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 3060 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 3120 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 3180 gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattca gcaaggtcgc 3240 cacgcacaag atcaatatta acaatcagtc atctctcttt agcaataaaa aggtgaaaaa 3300 ttacatttta aaaatgacac catagacgat gtatgaaaat aatctacttg gaaataaatc 3360 taggcaaaga agtgcaagac tgttacccag aaaacttaca aattgtaaat gagaggttag 3420 tgaagattta aatgaatgaa gatctaaata aacttataaa ttgtgagaga aattaatgaa 3480 tgtctaagtt aatgcagaaa cggagagaca tactatattc atgaactaaa agacttaata 3540 ttgtgaaggt atactttctt ttcacataaa tttgtagtca atatgttcac cccaaaaaag 3600 ctgtttgtta acttgtcaac ctcatttcaa aatgtatata gaaagcccaa agacaataac 3660 aaaaatattc ttgtagaaca aaatgggaaa gaatgttcca ctaaatatca agatttagag 3720 caaagcatga gatgtgtggg gatagacagt gaggctgata aaatagagta gagctcagaa 3780 acagacccat tgatatatgt aagtgaccta tgaaaaaaat atggcatttt acaatgggaa 3840 aatgatgatc tttttctttt ttagaaaaac agggaaatat atttatatgt aaaaaataaa 3900 agggaaccca tatgtcatac catacacaca aaaaaattcc agtgaattat aagtctaaat 3960 ggagaaggca aaactttaaa tcttttagaa aataatatag aagcatgcca tcatgacttc 4020 agtgtagaga aaaatttctt atgactcaaa gtcctaacca caaagaaaag attgttaatt 4080 agattgcatg aatattaaga cttattttta aaattaaaaa accattaaga aaagtcaggc 4140 catagaatga cagaaaatat ttgcaacacc ccagtaaaga gaattgtaat atgcagatta 4200 taaaaagaag tcttacaaat cagtaaaaaa taaaactaga caaaaatttg aacagatgaa 4260 agagaaactc taaataatca ttacacatga gaaactcaat ctcagaaatc agagaactat 4320 cattgcatat acactaaatt agagaaatat taaaaggcta agtaacatct gtggcaatat 4380 tgatggtata taaccttgat atgatgtgat gagaacagta ctttacccca tgggcttcct 4440 ccccaaaccc ttaccccagt ataaatcatg acaaatatac tttaaaaacc attaccctat 4500 atctaaccag tactcctcaa aactgtcaag gtcatcaaaa ataagaaaag tctgaggaac 4560 tgtcaaaact aagaggaacc caaggagaca tgagaattat atgtaatgtg gcattctgaa 4620 tgagatccca gaacagaaaa agaacagtag ctaaaaaact aatgaaatat aaataaagtt 4680 tgaactttag ttttttttaa aaaagagtag cattaacacg gcaaagtcat tttcatattt 4740 ttcttgaaca ttaagtacaa gtctataatt aaaaattttt taaatgtagt ctggaacatt 4800 gccagaaaca gaagtacagc agctatctgt gctgtcgcct aactatccat agctgattgg 4860 tctaaaatga gatacatcaa cgctcctcca tgttttttgt tttcttttta aatgaaaaac 4920 tttatttttt aagaggagtt tcaggttcat agcaaaattg agaggaaggt acattcaagc 4980 tgaggaagtt ttcctctatt cctagtttac tgagagattg catcatgaat gggtgttaaa 5040 ttttgtcaaa tgctttttct gtgtctatca atatgaccgt gtgattttct tctttaacct 5100 gttgatggga caaattacgt taattgattt tcaaacgttg aaccaccctt acatatctgg 5160 aataaattct acttggttgt ggtgtatatt ttttgataca ttcttggatt ctttttgcta 5220 atattttgtt gaaaatgttt gtatctttgt tcatgagaga tattggtctg ttgttttctt 5280 ttcttgtaat gtcattttct agttccggta ttaaggtaat gctggcctag ttgaatgatt 5340 taggaagtat tccctctgct tctgtcttct gaaagagatt gtagaaagtt gatacaaaag 5400 ccgaattcg 5409 2 5546 DNA Artificial Sequence pPGM-2 vector 2 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 60 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 120 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 180 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 240 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 300 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 360 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg 420 ctaggtaata cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg 480 gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc cagcacagtg 540 gcggccgctc gagtctagag ggcccgttta aacacgcgtg tcgagaactt gtttattgca 600 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 660 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 720 ggcatgcaag ctggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 780 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 840 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat 900 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 960 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 1020 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 1080 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 1140 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 1200 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 1260 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 1320 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 1380 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 1440 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 1500 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 1560 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 1620 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 1680 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 1740 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 1800 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 1860 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 1920 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 1980 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2040 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 2100 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 2160 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 2220 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 2280 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2340 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 2400 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 2460 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 2520 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2580 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 2640 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 2700 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 2760 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2820 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2880 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2940 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3000 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3060 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3120 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 3180 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 3240 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 3300 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac 3360 gaattcagca aggtcgccac gcacaagatc aatattaaca atcagtcatc tctctttagc 3420 aataaaaagg tgaaaaatta cattttaaaa atgacaccat agacgatgta tgaaaataat 3480 ctacttggaa ataaatctag gcaaagaagt gcaagactgt tacccagaaa acttacaaat 3540 tgtaaatgag aggttagtga agatttaaat gaatgaagat ctaaataaac ttataaattg 3600 tgagagaaat taatgaatgt ctaagttaat gcagaaacgg agagacatac tatattcatg 3660 aactaaaaga cttaatattg tgaaggtata ctttcttttc acataaattt gtagtcaata 3720 tgttcacccc aaaaaagctg tttgttaact tgtcaacctc atttcaaaat gtatatagaa 3780 agcccaaaga caataacaaa aatattcttg tagaacaaaa tgggaaagaa tgttccacta 3840 aatatcaaga tttagagcaa agcatgagat gtgtggggat agacagtgag gctgataaaa 3900 tagagtagag ctcagaaaca gacccattga tatatgtaag tgacctatga aaaaaatatg 3960 gcattttaca atgggaaaat gatgatcttt ttctttttta gaaaaacagg gaaatatatt 4020 tatatgtaaa aaataaaagg gaacccatat gtcataccat acacacaaaa aaattccagt 4080 gaattataag tctaaatgga gaaggcaaaa ctttaaatct tttagaaaat aatatagaag 4140 catgccatca tgacttcagt gtagagaaaa atttcttatg actcaaagtc ctaaccacaa 4200 agaaaagatt gttaattaga ttgcatgaat attaagactt atttttaaaa ttaaaaaacc 4260 attaagaaaa gtcaggccat agaatgacag aaaatatttg caacacccca gtaaagagaa 4320 ttgtaatatg cagattataa aaagaagtct tacaaatcag taaaaaataa aactagacaa 4380 aaatttgaac agatgaaaga gaaactctaa ataatcatta cacatgagaa actcaatctc 4440 agaaatcaga gaactatcat tgcatataca ctaaattaga gaaatattaa aaggctaagt 4500 aacatctgtg gcaatattga tggtatataa ccttgatatg atgtgatgag aacagtactt 4560 taccccatgg gcttcctccc caaaccctta ccccagtata aatcatgaca aatatacttt 4620 aaaaaccatt accctatatc taaccagtac tcctcaaaac tgtcaaggtc atcaaaaata 4680 agaaaagtct gaggaactgt caaaactaag aggaacccaa ggagacatga gaattatatg 4740 taatgtggca ttctgaatga gatcccagaa cagaaaaaga acagtagcta aaaaactaat 4800 gaaatataaa taaagtttga actttagttt tttttaaaaa agagtagcat taacacggca 4860 aagtcatttt catatttttc ttgaacatta agtacaagtc tataattaaa aattttttaa 4920 atgtagtctg gaacattgcc agaaacagaa gtacagcagc tatctgtgct gtcgcctaac 4980 tatccatagc tgattggtct aaaatgagat acatcaacgc tcctccatgt tttttgtttt 5040 ctttttaaat gaaaaacttt attttttaag aggagtttca ggttcatagc aaaattgaga 5100 ggaaggtaca ttcaagctga ggaagttttc ctctattcct agtttactga gagattgcat 5160 catgaatggg tgttaaattt tgtcaaatgc tttttctgtg tctatcaata tgaccgtgtg 5220 attttcttct ttaacctgtt gatgggacaa attacgttaa ttgattttca aacgttgaac 5280 cacccttaca tatctggaat aaattctact tggttgtggt gtatattttt tgatacattc 5340 ttggattctt tttgctaata ttttgttgaa aatgtttgta tctttgttca tgagagatat 5400 tggtctgttg ttttcttttc ttgtaatgtc attttctagt tccggtatta aggtaatgct 5460 ggcctagttg aatgatttag gaagtattcc ctctgcttct gtcttctgaa agagattgta 5520 gaaagttgat acaaaagccg aattcg 5546 3 5601 DNA Artificial Sequence pPGM-3 vector 3 gaattcacta gtgattaggg cccgttgaca ttgattattg actagttatt aatagtaatc 60 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 240 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600 aagcagagct cgctagcggc cgcagatctg ttaactcgag aacttgttta ttgcagctta 660 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 720 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcggcat 780 gcaagctggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 840 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 900 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt 960 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 1020 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 1080 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 1140 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 1200 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 1260 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 1320 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 1380 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 1440 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 1500 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 1560 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1620 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1680 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1740 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1800 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1860 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1920 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1980 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 2040 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 2100 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 2160 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 2220 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 2280 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 2340 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 2400 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 2460 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 2520 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 2580 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2640 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 2700 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2760 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2820 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2880 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2940 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 3000 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 3060 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 3120 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 3180 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 3240 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 3300 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 3360 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt 3420 cagcaaggtc gccacgcaca agatcaatat taacaatcag tcatctctct ttagcaataa 3480 aaaggtgaaa aattacattt taaaaatgac accatagacg atgtatgaaa ataatctact 3540 tggaaataaa tctaggcaaa gaagtgcaag actgttaccc agaaaactta caaattgtaa 3600 atgagaggtt agtgaagatt taaatgaatg aagatctaaa taaacttata aattgtgaga 3660 gaaattaatg aatgtctaag ttaatgcaga aacggagaga catactatat tcatgaacta 3720 aaagacttaa tattgtgaag gtatactttc ttttcacata aatttgtagt caatatgttc 3780 accccaaaaa agctgtttgt taacttgtca acctcatttc aaaatgtata tagaaagccc 3840 aaagacaata acaaaaatat tcttgtagaa caaaatggga aagaatgttc cactaaatat 3900 caagatttag agcaaagcat gagatgtgtg gggatagaca gtgaggctga taaaatagag 3960 tagagctcag aaacagaccc attgatatat gtaagtgacc tatgaaaaaa atatggcatt 4020 ttacaatggg aaaatgatga tctttttctt ttttagaaaa acagggaaat atatttatat 4080 gtaaaaaata aaagggaacc catatgtcat accatacaca caaaaaaatt ccagtgaatt 4140 ataagtctaa atggagaagg caaaacttta aatcttttag aaaataatat agaagcatgc 4200 catcatgact tcagtgtaga gaaaaatttc ttatgactca aagtcctaac cacaaagaaa 4260 agattgttaa ttagattgca tgaatattaa gacttatttt taaaattaaa aaaccattaa 4320 gaaaagtcag gccatagaat gacagaaaat atttgcaaca ccccagtaaa gagaattgta 4380 atatgcagat tataaaaaga agtcttacaa atcagtaaaa aataaaacta gacaaaaatt 4440 tgaacagatg aaagagaaac tctaaataat cattacacat gagaaactca atctcagaaa 4500 tcagagaact atcattgcat atacactaaa ttagagaaat attaaaaggc taagtaacat 4560 ctgtggcaat attgatggta tataaccttg atatgatgtg atgagaacag tactttaccc 4620 catgggcttc ctccccaaac ccttacccca gtataaatca tgacaaatat actttaaaaa 4680 ccattaccct atatctaacc agtactcctc aaaactgtca aggtcatcaa aaataagaaa 4740 agtctgagga actgtcaaaa ctaagaggaa cccaaggaga catgagaatt atatgtaatg 4800 tggcattctg aatgagatcc cagaacagaa aaagaacagt agctaaaaaa ctaatgaaat 4860 ataaataaag tttgaacttt agtttttttt aaaaaagagt agcattaaca cggcaaagtc 4920 attttcatat ttttcttgaa cattaagtac aagtctataa ttaaaaattt tttaaatgta 4980 gtctggaaca ttgccagaaa cagaagtaca gcagctatct gtgctgtcgc ctaactatcc 5040 atagctgatt ggtctaaaat gagatacatc aacgctcctc catgtttttt gttttctttt 5100 taaatgaaaa actttatttt ttaagaggag tttcaggttc atagcaaaat tgagaggaag 5160 gtacattcaa gctgaggaag ttttcctcta ttcctagttt actgagagat tgcatcatga 5220 atgggtgtta aattttgtca aatgcttttt ctgtgtctat caatatgacc gtgtgatttt 5280 cttctttaac ctgttgatgg gacaaattac gttaattgat tttcaaacgt tgaaccaccc 5340 ttacatatct ggaataaatt ctacttggtt gtggtgtata ttttttgata cattcttgga 5400 ttctttttgc taatattttg ttgaaaatgt ttgtatcttt gttcatgaga gatattggtc 5460 tgttgttttc ttttcttgta atgtcatttt ctagttccgg tattaaggta atgctggcct 5520 agttgaatga tttaggaagt attccctctg cttctgtctt ctgaaagaga ttgtagaaag 5580 ttgatacaaa agccgaattc g 5601 4 20 DNA Artificial Sequence primer for MAR of chicken lysozyme A 4 ggatccataa tataactgta 20 5 20 DNA Artificial Sequence primer for MAR of chicken lysozyme A 5 aagcttaaaa gattgaagca 20 6 20 DNA Artificial Sequence primer for chicken phi alpha-globin 5′ MAR 6 aagcttttaa ccaacaaaaa 20 7 20 DNA Artificial Sequence primer for chicken phi alpha-globin 5′ MAR 7 ctgcagacct aacctgtcac 20 8 20 DNA Artificial Sequence primer for CHO DHFR intron MAR 8 tatacgtgaa tagtttttct 20 9 20 DNA Artificial Sequence primer for CHO DHFR intron MAR 9 gagttggaac tgagaagttc 20 10 20 DNA Artificial Sequence primer for human HPRT intron MAR 10 aagcttggtc aagaatgctg 20 11 20 DNA Artificial Sequence primer for human HPRT intron MAR 11 gctgggcgtg gtggtgcctg 20 12 20 DNA Artificial Sequence primer for human CSP-B gene flanking SAR element 12 ggatcccatt ctccttgatg 20 13 20 DNA Artificial Sequence primer for human CSP-B gene flanking SAR element 13 gaattcaaac aactcaatag 20 14 20 DNA Artificial Sequence primer for human interferon-beta gene flanking SAR element 14 gaattcagca aggtcgccac 20 15 20 DNA Artificial Sequence primer for human interferon-beta gene flanking SAR element 15 ttgtatcaac tttctacaat 20 16 31 DNA Artificial Sequence sense primer for constructing pSV-beta-gal /ver1 vector 16 gcactagtcc cgggcccatg attacgaatt c 31 17 25 DNA Artificial Sequence sense primer for constructing pSV-beta-gal /ver1 vector 17 gtgccagctt gcatgcctgc aggtc 25 18 24 DNA Artificial Sequence primer 18 agggcccgtt gacattgatt attg 24 19 36 DNA Artificial Sequence primer 19 aagcttgcta gcgagctctg cttatataga cctccc 36 20 26 DNA Artificial Sequence primer 20 cctaggtaat acgactcact ataggg 26 21 31 DNA Artificial Sequence primer 21 gtcgacacgc gtgtttaaac gggccctcta g 31 

What is claimed is:
 1. A mammalian expression vector comprising: (a) a nuclear matrix attachment region of interferon β gene; (b) a promoter; and (c) a transcription terminator.
 2. The mammalian expression vector according to claim 1, wherein the promoter is selected from a group consisting of SV40 promoter, CMV (cytomegalovirus) promoter, and MMTV (mouse mammary tumor virus) promoter.
 3. The mammalian expression vector according to claim 1, wherein the mammalian expression vector is pPGM-1 KCCM
 10232. 4. The mammalian expression vector according to claim 1, wherein the mammalian expression vector is pPGM-2 KCCM
 10338. 5. The mammalian expression vector according to claim 1, wherein the mammalian expression vector is pPGM-3 KCCM
 10339. 6. A cell transfected with a mammalian expression vector of claim 1 in which target gene was inserted.
 7. The cell according to claim 6, wherein the mammalian expression vector is selected from a group consisting of pPGM-1 (KCCM 10232), pPGM-2 (KCCM 10338), and pPGM-3 (KCCM 10339).
 8. The cell according to claim 6, wherein the cell is a mammalian cell or a cell derived from a mammalian organism.
 9. The cell according to claim 6, wherein the cell is derived from CHO (Chinese hamster ovary) cell line.
 10. A method of producing recombinant protein comprising: introducing a transgene into the mammalian expression vector of claim 1; and transfecting mammalian cells with the expression vector in order to express protein from the transgene.
 11. The method according to claim 10, wherein the mammalian expression vector is selected from a group consisting of pPGM-1 (KCCM 10232), pPGM-2 (KCCM 10338), and pPGM-3 (KCCM 10339). 